Chromosomal rearrangements are a common genomic abnormality found in tumor cells, which can alter the normal function of the genes involved, thus contributing to cancer development. Despite their prevalence, much remains unknown about the mechanisms that form translocations. Several commonly occurring chromosomal rearrangements, RET/PTC, AKAP9/BRAF, STRN/ALK, ETV6/NTRK3 and several NTRK1 gene fusions, contribute to the development of papillary thyroid carcinomas (PTC), a major type of thyroid cancer. In the US general population, thyroid cancer is the fastest increasing type of cancer. Its incidence has increased 4 to 7-fold in US Air Force active-duty personnel compared to the general population, even though overall cancers are less frequent in military personnel. We have demonstrated for the first time that DNA breaks at chromosomal fragile sites participate directly in the generation of oncogenic RET/PTC1 rearrangements in human thyroid cells. Fragile sites are sensitive to a range of chemicals, and have been identified in the regions of deletions and chromosomal rearrangements. All partner genes participating in PTC rearrangements are located in known fragile sites. Several environmental exposures, which are daily encountered by the general population and/or intensified in military personnel, such as benzene (in explosives, jet and automobile exhausts, and napalm) and diethylnitrosamine (in cigarette smoke and pesticides), also induce fragile site breakage. Many of them, including benzene, show a positive association with the risk of thyroid cancer. We hypothesize that these agents alone or in combination contribute to the increased incidence of thyroid cancer. In this proposal, we will investigate whether environmental exposures generate RET fragility, leading to RET/PTC rearrangements in human thyroid cells by examining a variety of environmental and therapeutic agents known to induce fragile sites, and will expand this study to other PTC-specific rearrangements as well. Next, we will identify the mechanism of fragile site-mediated RET/PTC rearrangements in response to these environmental exposures, by determining whether the formation of secondary structures during DNA replication contribute to RET gene instability in the formation of RET/PTC rearrangements. Then, to work towards clinical application of DNA fragility to a DNA diagnostic test, we will test whether breakage of RET and other rearrangement-participating genes in normal cells of PTC patients with rearrangements is higher than that in normal individuals, as a means to evaluate individual susceptibility to PTC. This proposal will have significant impact on our understanding of the direct role of environmental factors in the RET/PTC and other rearrangements of thyroid cancers. These mechanistic studies will provide new knowledge to better understand fragile site breakage and its role in sporadic cancer initiation. Further, using data on preferential breakage properties of rearrangement-participating genes to create a DNA test can potentially be extended to other cancers caused by fragile site-mediated rearrangements.